Long-chain fatty acids (FA) have multiple properties and functions. FA are important substrates for phospholipids, which are essential membrane components. FA are also substrates for prostaglandins, which have a variety of regulatory effects. For most cells FA constitute a main source of energy. FA also directly regulate a variety of biological processes. For example, FA modulate ion channel activation, enzyme function and synaptic transmission. More recently FA have been shown to have regulatory effects on the expression of various genes, especially those encoding proteins active in lipid metabolism.
In view of the diverse functionality of FA, researchers believe that FA play a central role in the pathophysiology of multiple conditions, such as diabetes and obesity, for example. FA also contribute to insulin insensitivity and to the prevalence of vascular and coronary diseases. High levels of circulating blood FA are also associated with diabetes, many forms of obesity and hyperlipidemias. High levels of blood FA are believed to contribute to an increased production of low-density lipoproteins by the liver. Cholesterol from lipoproteins is esterified with free FA by macrophages in the vascular wall yielding cholesteryl ester which accumulates and leads to the formation of lipid-filled macrophages, precursors of atherosclerotic lesions.
The wide range of effects and physiological functions of FA underscore the importance of understanding how cellular FA uptake is regulated.
The mechanism of FA transfer across cell membranes has long been postulated to occur by simple diffusion. However, most recent biophysical studies indicate that FA diffusion may not be fast enough to accommodate FA uptake by cells active in FA metabolism. Furthermore, FA circulate in blood tightly bound to serum albumin which markedly limits FA partition into membrane lipid. Therefore the quantity of free FA that is available for cellular uptake is extremely low, i.e. in the nanomolar range.
Ibrahimi, et al. (1996) Proc. Natl. Acad. Sci. USA 93:2646-2651, provide biochemical evidence to support the involvement of a membrane carrier which mediates cellular uptake of long chain FA. Specifically, an 88-kDa membrane protein has been identified and isolated. This high-affinity long-chain FA transporter isolated from mice is highly homologous to human CD36. Recently, Abumrad, et al. (1993) J. Biol. Chem. 268(24):17665-17668, showed that CD36 was highly expressed in tissues active in FA utilization such as the heart, adipose tissue and intestine. CD36 is absent from the brain which does not utilize long-chain FA. CD36 is highly expressed in red oxidative muscle but not in white glycolytic muscle. CD36 is upregulated during muscle development and muscle stimulation when FA utilization increases, (see, Sfeir, et al. (1997) Prostaglandins, Leukotrienes and Essential Fatty Acids 57(1):17-21).
Han, et al. (1997) J. Biol. Chem. 272(34):21654-21659 studied the impact of lipids on the expression of CD36 and found that low density lipoproteins induced CD36 expression in a murine macrophage cell line. Han, et al. suggest the use of "knockout" mice that lack the expression of CD36 to assess the in vivo function of CD36 receptors.
Van Nieuwenhoven, et al. (1995) Biochem. and Biophys. Res. Comm. 207(2):747-752 report expression of CD36 in muscle tissue and cell types with high fatty acid metabolism and suggest a role of CD36 in fatty acid metabolism when co-expressed with another protein known as cytoplasmic fatty acid-binding protein (FABP).
Until now, there have been no satisfactory animal models in which tissue-specific overexpression of a protein and concomitant increased transport of FA can be made to occur in a reliable and predictable fashion in a substantial proportion of animals.
The mouse model of the present invention can be reliably and predictably used to assess whether overexpression of CD36 in muscle can reverse hyperlipidemias, decrease obesity, improve insulin sensitivity and lower the risk of atherosclerosis.